The present invention is in the field of secreted proteins that are related to the transcobalamin II secreted subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
Secreted Proteins
Many human proteins serve as pharmaceutically active compounds. Several classes of human proteins that serve as such active compounds include hormones, cytokines, cell growth factors, and cell differentiation factors. Most proteins that can be used as a pharmaceutically active compound fall within the family of secreted proteins. It is, therefore, important in developing new pharmaceutical compounds to identify secreted proteins that can be tested for activity in a variety of animal models. The present invention advances the state of the art by providing many novel human secreted proteins.
Secreted proteins are generally produced within cells at rough endoplasmic reticulum, are then exported to the golgi complex, and then move to secretory vesicles or granules, where they are secreted to the exterior of the cell via exocytosis.
Secreted proteins are particularly useful as diagnostic markers. Many secreted proteins are found, and can easily be measured, in serum. For example, a xe2x80x98signal sequence trapxe2x80x99 technique can often be utilized because many secreted proteins, such as certain secretory breast cancer proteins, contain a molecular signal sequence for cellular export. Additionally, antibodies against particular secreted serum proteins can serve as potential diagnostic agents, such as for diagnosing cancer.
Secreted proteins play a critical role in a wide array of important biological processes in humans and have numerous utilities; several illustrative examples are discussed herein. For example, fibroblast secreted proteins participate in extracellular matrix formation. Extracellular matrix affects growth factor action, cell adhesion, and cell growth. Structural and quantitative characteristics of fibroblast secreted proteins are modified during the course of cellular aging and such aging related modifications may lead to increased inhibition of cell adhesion, inhibited cell stimulation by growth factors, and inhibited cell proliferative ability (Eleftheriou et al., Mutat Res 1991 March-November; 256(2-6): 127-38).
The secreted form of amyloid beta/A4 protein precursor (APP) functions as a growth and/or differentiation factor. The secreted form of APP can stimulate neurite extension of cultured neuroblastoma cells, presumably through binding to a cell surface receptor and thereby triggering intracellular transduction mechanisms. (Roch et al., Ann N Y Acad Sci Sep. 24, 1993;695:149-57). Secreted APPs modulate neuronal excitability, counteract effects of glutamate on growth cone behaviors, and increase synaptic complexity. The prominent effects of secreted APPs on synaptogenesis and neuronal survival suggest that secreted APPs play a major role in the process of natural cell death and, furthermore, may play a role in the development of a wide variety of neurological disorders, such as stroke, epilepsy, and Alzheimer""s disease (Mattson et al., Perspect Dev Neurobiol 1998; 5(4):337-52).
Breast cancer cells secrete a 52K estrogen-regulated protein (see Rochefort et al., Ann N Y Acad Sci 1986;464:190-201). This secreted protein is therefore useful in breast cancer diagnosis.
Two secreted proteins released by platelets, platelet factor 4 (PF4) and beta-thromboglobulin (betaTG), are accurate indicators of platelet involvement in hemostasis and thrombosis and assays that measure these secreted proteins are useful for studying the pathogenesis and course of thromboembolic disorders (Kaplan, Adv Exp Med Biol 1978;102:105-19).
Vascular endothelial growth factor (VEGF) is another example of a naturally secreted protein. VEGF binds to cell-surface heparan sulfates, is generated by hypoxic endothelial cells, reduces apoptosis, and binds to high-affinity receptors that are up-regulated by hypoxia (Asahara et al., Semin Interv Cardiol 1996 September;1(3):225-32).
Many critical components of the immune system are secreted proteins, such as antibodies, and many important functions of the immune system are dependent upon the action of secreted proteins. For example, Saxon et al., Biochem Soc Trans 1997 May;25(2):383-7, discusses secreted IgE proteins.
For a further review of secreted proteins, see Nilsen-Hamilton et al., Cell Biol Int Rep 1982 September;6(9):815-36.
Transcobalamin II
Many biochemical reactions require the involvement of cobalamin (xe2x80x9cCblxe2x80x9d), also known as vitamin B12, as coenzyme factors. Human Cbl-dependent metabolism includes the biosynthesis of methionine from homocysteine and the isomerization of methylmalonyl-CoA to succinyl-CoA. Although cobalamin is highly water-soluble, it is nevertheless impervious to plasma membrane. Cobalamin is delivered into the designated subcellular locations through multiple physiological steps.
The cellular uptake of cobalamin is mediated by transcobalamin II (TCII), a plasma protein that binds Cbl and is secreted by human umbilical vein endothelial (HUVE) cells. These cells synthesize and secrete TC II and, therefore, served as the source of the library from which the TC II cDNA was isolated. This full-length cDNA consists of 1866 nucleotides that code for a leader peptide of 18 amino acids, a secreted protein of 409 amino acids, a 5xe2x80x2-untranslated segment of 37 nucleotides, and a 3xe2x80x2-untranslated region of 548 nucleotides. A single 1.9-kilobase species of mRNA corresponding to the size of the cDNA was identified by Northern blot analysis of the RNA isolated from HUVE cells. TCII has 20% amino acid homology and greater than 50% nucleotide homology with human transcobalamin I (TCI) and with rat intrinsic factor (R-IF). TCII has no homology with the amino-terminal region of R-IF that has been reported to have significant primary as well as secondary structural homology with the nucleotide-binding domain of NAD-dependent oxidoreductases. The regions of homology that are common to all three proteins are located in seven domains of the amino acid sequence. One or more of these conserved domains is likely to be involved in Cbl binding, a function that is common to all three proteins. However, the difference in the affinity of TCII, TCI, and R-IF for Cbl and Cbl analogues indicates, a priori, that structural differences in the ligand-binding site of these proteins exist and these probably resulted from divergence of a common ancestral gene. (Platica, et al., J Biol Chem Apr 25;266(12):7860-3 (1991))
Extracellularly secreted cobalamin is continually transported across cellular space through transcytosis within the endomembrane-secretory system, within which transcobalamin II (xe2x80x9cTC IIxe2x80x9d) binds to proteolytically-released Cbl. TC II-Cbl-containing vesicles release their contents into the circulation system. The uptake of TC II-Cbl from the circulating fluids utilizes similar pathways, including receptor-mediated translocation, vesicle-dependent trafficking and targeting, and lysosome-based proteolytical release.
TC II is a non-glycosylated secretory protein of molecular mass 43 kDa in plasma while its homologs IF and haptocorrin are heavily glycosylated. A conserved Cbl-binding domain (ProSite pattern: PS000468) exists among the three types of the proteins (Seetharam and Li, Vitam. Horm. 59:337-366 (2000); Seetharam B, et al., Annu. Rev. Nutr. 19:173-195 (1999); Hofman, et al., Nuc. Acid Res. 27: 215-219 (1999)). The affinity toward Cbl is suggested to be the highest for haptocorrin (Fedosov, et al., Biochim. Biophys. Acta 1292:113-119 (1996)). Of two TCs, TC I has been identified as a major protein constituent of secondary granules in neotrophil and mapped onto chromosome 11q11-q12 (Johnston, et al., J. Biol. Chem. 264:15754-15757 (1989)). IF (Chr 11), TC I and TC II (Chr 22q) are proposed to be diverged from a common ancestral gene as they are conserved in the multiple regions, but with different affinity toward Cbl (Platica, et al., J Biol Chem Apr 25;266(12):7860-3 (1991)).
Disorders of transport proteins such as TC II can lead to abnormal function of methylmalonyl-CoA mutase and methionine synthase (Fowler, Eur. J. Pediatr. 157(suppl. 2):S60-S66 (1998)). Clinical evidence has demonstrated that autosomal recessive mutations of TC II gene can lead to a disorder whose observed symptoms include megaloblastic anemia, impaired immune response and neurological manifestations. Li, et al., Hum. Mol. Genet. 3:1835-1840 (1994). Single nucleotide deletions in patients were reported to cause TC II deficiency disease. (Li, et al., Biochem. Biophys. Res. Commun. 204:1111-1118 (1994)).
Cancer cells are commonly characterized by a disturbed balance of methionine metabolism, resulting in ceased proliferation of methionine-dependent cells and over-production of methionine-independent cells. The imbalance between methionine comsumption and formation is related to methionine synthase and methylcobalamin cofactor. The lack of cellular methylcobalamin, resulted from various defects in cobalamin metabolism as depicted above, causes a low rate of homocysteine remethylation, and thus methionine production (Fiskerstrand, et al., J. Biol. Chem. 273: 20180-20184 (1998)).
Secreted proteins, particularly members of the transcobalamin II secreted protein subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of secreted proteins. The present invention advances the state of the art by providing previously unidentified human secreted proteins that have homology to members of the transcobalamin II secreted protein subfamily.
The present invention is based in part on the identification of amino acid sequences of human secreted peptides and proteins that are related to the transcobalamin II secreted protein subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate secreted protein activity in cells and tissues that express the secreted protein. Experimental data as provided in FIG. 1 indicates expression of isoform 1 in adult adrenal gland, mammary gland, retinoblastoma, adenocarcinoma cell line, embryonal carcinoma cell line, adult uterus, adult head-neck, and leukocytes. Experimental data as provided in FIG. 1 indicates expression of isoform 2 in adult adrenal gland, adult uterus, adult head-neck, adult lung tumor, mammary gland, retinoblastoma, adenocarcinoma, and the hippocampus.